Transcription terminators are sequences in DNA that stop the progress of RNA polymerase and thereby reduce the transcription of downstream genes. Antiterminators can increase downstream gene expression by modifying polymerase so that it no longer recognizes or responds to terminators. Nascent transcripts encoded by the cis acting antitermination sites ("put" sites) of bacteriophage HK022 promote such a modification: an E. coli RNA polymerase molecule that transcribes a put site acquires an increased ability to transcribe through downstream transcription terminators. Antitermination is required for the expression of most phage genes. Put suppresses both factor dependent and intrinsic terminators and has no apparent terminator specificity. Efficient antitermination depends on the structure of the put transcript and of a highly conserved Zn-finger motif located in beta', the largest subunit of RNA polymerase. No additional protein factor is required. The Zn-finger motif contains two pairs of invariant cysteines flanking a moderately well conserved segment of 13 amino acids that is rich in basic residues. To investigate the role of this region in enzyme function, we replaced each of the charged residues with alanine and determined the effects of the substitutions on cell phenotype and enzymatic activity. All of the mutated genes complemented a temperature sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro. Therefore, the mutant enzymes are competent to perform essential cellular functions. However, none of the mutants antiterminated transcription with normal efficiency in response to phage HK022 put sites. We found that the severity of the antitermination defect depended on the sequence of the upstream stem-loop of put RNA. Some but not all Zn-finger mutants could distinguish between put variants that differed in this region. This mutant specificity suggests that put RNA interacts directly and specifically with the Zn-finger motif to effect antitermination. This conclusion is supported by our recent finding that one of the Zn-finger mutants fails to bind nascent put RNA during transcription in vitro. Substitution or deletion of the invariant cysteines differs from substitution of the charged residues in that the cysteine mutants are unable to complement a temperature sensitive beta' mutant for cell growth. The RNA polymerase produced by these mutants is active in transcription but terminates less efficiently than the wild type enzyme on templates that lack a put site. In addition, the mutant enzymes do not respond to put sites. It therefore appears that the Zn-finger motif has a role in termination as well as in binding put RNA. These two properties could well be related: binding of put RNA by wild type polymerase could alters the structure of the Zn-finger motif in the same way as mutations of the invariant cysteines.